The goals of this research are: to determine how myoblasts and their mesodermal precursors come to be spatially ordered in discrete regions in the developing vertebrate limb, and to determine how removal of specific mitrogens results in the withdrawal of myoblasts from proliferation, and in their commitment to terminal muscle differentiation. The problem of spatial ordering is approached using in vitro clonal assays for muscle colony-forming cells (MCF cells), and a procedure for cutting 30-300Mu sections through living limb buds. With this approach we will: (a) complete a descriptive study of MCF cell distribution in chick wing and leg buds, and in comparable mouse and human stages; and (b) determine how (in chick limb buds) MCF cell gradients are affected by manipulating various limb tissues. In addition, we will: compare the protein species being synthesized by prospective myogenic and chondrogenic regions; compare early and late MCF cells with respect to myosin light chain synthesis, and antigenic differences detected by monoclonal antisera, and investigate factors required for the in vitro conversion of early myogenic precursors to MCF cells. The problem of how mitogen removal regulates myoblast commitment to terminal differentiation is approached using permanent clonal mouse myoblast cell lines and a series of mutants derived from these. The project will involve: (a) defining the range of mitrogens to which mouse myoblasts respone, (b) isolating new clonal myogenic cell lines which differ in their mitogen responsiveness; (c) determining how myoblasts remove or destroy mitrogens in their environment; (d) determining when, during the cell cycle, myoblasts commit to terminal differentiation in response to mitogen removal; analyzing sequential biochemical changes, accompanying the commitment of myoblasts to terminal differentiation; (f) determining how, at the gene and chromosomal level, commitment is inhibited by bromodeoxyuridine; (g) determining the "rules" by which a committed nucleus affects non-committed nuclei in various heterokaryon combinations; (h) isolating a series of temperature-sensitive, commitment-negative myoblast mutants and using these to genetically dissect the commitment mechanisms; and (i) isolating muscle-specific gene expression mutants which will be used to analyze how muscle genes are regulated during terminal differentiation.